Small RNAs Do It Better

At the beginning of the 21st century, the Human Genome Project revealed that, in contrast to the expected 100,000, only 20,000 to 25,000 protein-coding genes were present, accounting for 2% of the human genome.1International Human Genome Sequencing ConsortiumFinishing the euchromatic sequence of the human genome.Nature. 2004; 431: 931-945Google Scholar A tiny number compared with that of organisms such as Arabidopsis thaliana (25,000 genes) or Caenorhabditis elegans (20,000 genes) and certainly not enough to explain the complexity, diversity, and evolution of human beings. Therefore, 98% of the human genome was labeled as “junk DNA.” Soon after, with the widespread application of high-throughput sequencing, either in bulk or on single cells, abundant noncoding RNAs were detected and at present it is estimated that up to 80% of the human genome has the capacity to transcribe into noncoding RNA with complex regulatory dynamics.2Ecker J.R. Bickmore W.A. Barroso I. Pritchard J.K. Gilad Y. Segal E. Genomics: ENCODE explained.Nature. 2012; 489: 52-55Google Scholar noncoding RNA are a precious resource for the genome as they modulate complex molecular and cellular processes and are key players in gene-regulatory networks, a scenario that better reflects the human evolutionary identity. The advent of liquid biopsy for minimally invasive cancer detection and treatment monitoring has prompted the search for informative biomarkers in blood and other body fluids. So far, most of the multicancer early detection tests that reached an advanced validation phase have been on the basis of the detection of different features of circulating tumor DNA including gene mutation signatures,3Lennon A.M. Buchanan A.H. Kinde I. et al.Feasibility of blood testing combined with PET-CT to screen for cancer and guide intervention.Science. 2020; 369eabb9601Google Scholar fragmentomics,4Mathios D. Johansen J.S. Cristiano S. et al.Detection and characterization of lung cancer using cell-free DNA fragmentomes.Nat Commun. 2021; 12: 5060Google Scholar methylation,5Klein E.A. Richards D. Cohn A. et al.Clinical validation of a targeted methylation-based multi-cancer early detection test using an independent validation set.Ann Oncol. 2021; 32: 1167-1177Google Scholar and circulating tumor cells.6Marquette C.H. Boutros J. Benzaquen J. et al.AIR project Study Group. Circulating tumour cells as a potential biomarker for lung cancer screening: a prospective cohort study.Lancet Respir Med. 2020; 8: 709-716Google Scholar However, when we look at early phases of cancer development or, even more crucially, at risk prediction, markers signaling strictly tumor-related changes could be a limiting factor, as indicated by the low stage-dependent sensitivity of these assays. This is particularly true in screening programs for lung cancer (LC), in which at risk but cancer-free individuals are enrolled. Instead, the approach of interrogating not only the tumor but also its micro-macroenvironment and the patient’s whole body (including the immune system) is meaningful and much more promising. Small RNA molecules (sRNAs), including microRNAs (miRNAs) and other species (transfer RNAs, ribosomal RNA [rRNAs], small nucleolar RNAs) carrying information across many biological phenotypes may better inform about cancer risk and early cancer interception in LC screening trials. In this issue of the Journal of Thoracic Oncology, Sikosek et al.7Sikosek T. Horos R. Trudzinski F. et al.Early detection of lung cancer using small RNAs.J Thorac Oncol. 2023; 18: 1504-1523Google Scholar reports on the development of a diagnostic model on the basis of sRNAs in large multicenter cohorts (seven centers) from Germany and the United States. To mimic a real-world LC screening cohort, individuals were enrolled on the basis of the National Lung Screening Trial eligibility criteria for LC (age 55–75 y; smoking history ≥30 pack-year), and whole blood was collected either at diagnosis of confirmed LC (cases) or after ruling out LC (controls). Interestingly, the authors used whole-blood samples to run ultradeep sRNA sequencing (20 million reads per sample) coupled with a preanalytical method to remove highly abundant erythroid RNAs to account for the massive hemolysis and to allow the detection of less abundant species released by the tumor and the immune-cell compartment. By using an accurate bioinformatic pipeline, they discovered in the training set of 943 individuals (445 LC, 498 no LC) an 18-sRNA feature consensus signature (50% miRNAs) termed miLung, which was validated in an independent cohort of 441 individuals (286 LC, 155 no LC). Their diagnostic model revealed remarkable operational characteristics, with receiver operating characteristic area under the curve values of 0.86 and 0.83 in the discovery and validation sets, respectively. Its performance decreased significantly in stage I LC (0.73 and 0.76) while reaching area under the curve values of 0.90 and 0.86 in stage IV. The detection of squamous cell carcinoma was better (0.88) than that of lung adenocarcinoma (LUAD) (0.81) and peaked in SCLC (0.93, 0.91). In a subset of 40 individuals, blood-cell sorting, and tumor-tissue sequencing were performed to deconvolve sRNAs into their source of origin. Deconvolution of the miLung signature in this parallel subset of samples revealed that 39% of the feature source was composed of plasma, whereas erythrocytes and thrombocytes still represented a large fraction (28.6%) and the remaining 32.4% were immune-cell subtypes. The most highly weighted feature in the model was an rRNA fragment, 28S-rRNA-bin-162, originating exclusively from the plasma fraction of whole blood. These findings are in keeping with the concept that most informative biomarkers are able to amplify the early cancer signal derived from the integration of both tumor and host-derived information. An additional experiment using dried capillary blood spot collection and sequencing in 19 patients with early-stage LC and 21 controls revealed a strong correlation among miLung signatures in whole blood and capillary blood, opening up the prospect of noninvasive home-sampling of dried blood. This study adds further evidence on the use of circulating-sRNA–based tests as biomarkers for early detection and screening of LC. Previous studies have pointed out the efficacy of this class of molecules, particularly circulating miRNAs. Since 2011, we have been pioneering a blood miRNA assay to improve the performance of low-dose computed tomography (LDCT) screening and we have released an miRNA signature classifier (MSC)8Boeri M. Verri C. Conte D. et al.MicroRNA signatures in tissues and plasma predict development and prognosis of computed tomography detected lung cancer.Proc Natl Acad Sci U S A. 2011; 108: 3713-3718Google Scholar,9Sozzi G. Boeri M. Rossi M. et al.Clinical utility of a plasma-based microRNA signature classifier within computed tomography lung cancer screening: a correlative MILD trial study.J Clin Oncol. 2014; 32: 768-773Google Scholar to be tested in the BioMILD trial, a prospective single-arm LDCT screening study with 4119 participants.10Pastorino U. Boeri M. Sestini S. et al.Baseline computed tomography screening and blood microRNA predict lung cancer risk and define adequate intervals in the BioMILD trial.Ann Oncol. 2022; 33: 395-405Google Scholar The baseline CT and MSC results were used up front to choose the screening intervals, and participants with a double-negative LDCT/MSC result underwent repeat LDCT after 3 years. The BioMILD results at 5.5 years revealed a major predictive value of MSC positivity for LC incidence and mortality in CT-indeterminate/positive participants, with no risk of late LC detection. In contrast, the added value of MSC in LDCT-negative participants was low, and the number of false-positive MSC results was high. In agreement with the study by Sikosek et al.,7Sikosek T. Horos R. Trudzinski F. et al.Early detection of lung cancer using small RNAs.J Thorac Oncol. 2023; 18: 1504-1523Google Scholar we noticed that most of the 24 miRNAs composing the MSC were expressed and released in the culture medium by hematopoietic lineages and other stromal cells such as lung fibroblasts and endothelial cells.11Fortunato O. Borzi C. Milione M. et al.Circulating mir-320a promotes immunosuppressive macrophages M2 phenotype associated with lung cancer risk.Int J Cancer. 2019; 144: 2746-2761Google Scholar A screening strategy on the basis of the individual risk profile could have important implications for public health economics: reduction of costs and radiation exposure, improvement of long-term compliance, and a larger number of screened individuals. The advent of new ultralow-dose LDCT scanners and artificial intelligence programs, with an LC-negative predictive value greater than 98%, supports such a risk-based strategy. The team study by Sikosek et al.7Sikosek T. Horos R. Trudzinski F. et al.Early detection of lung cancer using small RNAs.J Thorac Oncol. 2023; 18: 1504-1523Google Scholar has several major strengths. First, the analysis of large, prospectively collected multicenter cohorts selected with National Lung Screening Trial criteria, which somehow mimics the screening context. Second, the hypothesis-driven approach, consistent with the host-response concept on the basis of the integration of tumor- and host-derived information, facilitates the detection of early signals. Finally, the robust technological pipeline led to reduced hemolysis as a confounding factor and the use of whole blood and, potentially, dried capillary blood spots as starting material. A limitation of the study by Sikosek et al.7Sikosek T. Horos R. Trudzinski F. et al.Early detection of lung cancer using small RNAs.J Thorac Oncol. 2023; 18: 1504-1523Google Scholar is the case-control design focusing on prevalent LC, whereas the major issue in screening is incident and asymptomatic LC. Another limitation is the lack of association analyses with LDCT findings such as nodule size and radiomic features. Furthermore, the distribution of tumor stages and histologic subtypes was remarkably different from real-world screening data, with only 20.7% and 29% stage Ia plus b LC and 46.5% and 54.5% LUAD in the training and validation cohorts, respectively, compared with the greater than 60% and 70% stage I LC and LUAD observed in real-world screening. Unfortunately, the sensitivity of miLung was comparatively low in stage I LC and LUAD, the main targets of LC screening. Whereas the availability of an easy-to-use blood test may contribute to overcoming the well-known resistance of heavy smokers to LDCT screening, previous prospective studies with upfront blood biomarkers have reported the risk of false reassurance caused by a negative test with low sensitivity.12Sullivan F.M. Mair F.S. Anderson W. et al.Earlier diagnosis of lung cancer in a randomised trial of an autoantibody blood test followed by imaging.Eur Respir J. 2021; 572000670Google Scholar Therefore, it is essential to assess in a large-scale prospective trial involving high-risk individuals the real sensitivity of this sRNA test in stage I LC. Finally, the costs of ultradeep sequencing and the preanalytical blocking strategy could limit the accessibility of the test to communities with lower economic status and levels of education, in which smoking remains highly prevalent. But on the whole, this is a broad, innovative, and accurate study and we congratulate the authors on its sound results, which prompt a prospective LC screening validation trial to confirm the promise of the miLung classifier in a real-world screening setting. Gabriella Sozzi, Ugo Pastorino: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Validation, Visualization, Roles/Writing - original draft, Writing - review & editing. Early Detection of Lung Cancer Using Small RNAsJournal of Thoracic OncologyVol. 18Issue 11PreviewLung cancer remains the deadliest cancer in the world, and lung cancer survival is heavily dependent on tumor stage at the time of detection. Low-dose computed tomography screening can reduce mortality; however, annual screening is limited by low adherence in the United States of America and still not broadly implemented in Europe. As a result, less than 10% of lung cancers are detected through existing programs. Thus, there is a great need for additional screening tests, such as a blood test, that could be deployed in the primary care setting. Full-Text PDF Open Access